Neutrophils are the most abundant cell type among circulating white blood cells and constitute the first line of host defense against invading bacteria and other pathogens. They migrate toward sites of infection or inflammation by responding to gradients of chemoattractants, a process known as chemotaxis. Neutrophil chemotaxis is mediated by chemotactic signal transduction pathways. The long-term goal of this project is to elucidate the molecular basis of the chemotactic signaling. We are particularly interested in the signal pathway mediated by inositol phospholipid Ptdlns(3,4,5)P3. This pathway has proven to be essential for mediating chemotactic responses, while its regulation remains ill defined. Chemoattractant stimulation initiates localized accumulation of Ptdlns(3,4,5)P3 on the plasma membrane at the leading edge of chemotaxing cells. A set of pleckstrin homolog (PH) domain-containing proteins are then translocated onto the membrane via their specific binding to Ptdlns(3,4,5)P3 and subsequently trigger downstream signals leading to chemotaxis. The PH-domain membrane translocation was previously thought to be dependent solely upon concentrations of Ptdlns(3,4,5)P3 in the membrane. Recently, we discovered that an intracellular inositol phosphate, lns(1,3,4,5)P4, competes with Ptdlns(3,4,5)P3 for binding to the PH domain and attenuates PH-domain membrane translocation in neutrophils, providing a novel mode of regulation for PH domain function. This intriguing result led us to hypothesize that lns(1,3,4,5)P4, by suppressing PH- domain translocation, negatively regulates neutrophil chemotaxis. Consistent with this idea, our preliminary data show that lns(1,3,4,5)P4 level is greatly augmented during chemotaxis. In addition, treatment of neutrophils with membrane-permeant lns(1,3,4,5)P4 significantly inhibits their chemotactic movement. To further understand the regulation of Ptdlns(3,4,5)P3 signal by lns(1,3,4,5)P4 in chemotaxis, we will characterize the molecular mechanisms by which the intracellular level of lns(1,3,4,5)P4 is augmented by Chemoattractant stimulation (Aim I). Moreover, the physiological consequences of the chemoattractant- elicited augmentation of lns(1,3,4,5)P4 will be investigated using neutrophils lacking lns(1,3,4,5)P4 (Aim II). Finally, the contribution of lns(1,3,4,5)P4 to neutrophil chemotaxis in live animals will be investigated using a mouse peritonitis model and a dorsal air pouch model (Aim III). Together, these studies will provide a better understanding of the role of lns(1,3,4,5)P4 in neutrophil chemotaxis, with the ultimate goal of establishing lns(1,3,4,5)P4 and related pathways as novel therapeutic targets for modulating neutrophil functions. Accordingly, more efficient and effective therapies could be developed to treat a variety of infectious and inflammatory diseases, such as asthma, multiple sclerosis, and rheumatoid arthritis.